AU2019243093B2 - Nutritional composition, food/drink composition using nutritional composition, and modified milk powder using nutritional composition - Google Patents
Nutritional composition, food/drink composition using nutritional composition, and modified milk powder using nutritional composition Download PDFInfo
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- A23C9/20—Dietetic milk products not covered by groups A23C9/12 - A23C9/18
- A23C9/203—Dietetic milk products not covered by groups A23C9/12 - A23C9/18 containing bifidus-active substances, e.g. lactulose; containing oligosaccharides
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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Abstract
Provided is a technology that makes it possible to promote the growth, in the presence of human milk oligosaccharides (HMOs), of probiotics that have a low ability to assimilate HMOs. Provided is a nutritional composition that contains
Description
Title of Invention:
Technical Field
[0001]
The present technology relates to a nutritional composition
capable of promoting proliferation of probiotics having a low
ability to assimilate human milk oligosaccharides
(hereinafter, also referred to as "HMOs").
Background Art
[0002]
Bifidobacteria are indigenous bacteria within an intestinal
tract of mammals including humans, and are known to have an
action of inhibiting proliferation of pathogenic intestinal
bacteria within the intestinal tract.
In general, Bifidobacteria are dominant in intestinal flora
(intestinal bacteria flora) of a healthy babies, but gradually
decrease with age, and then putrefactive bacteria such as
Escherichiacoliorbacteriaof the genusClostridiumincrease,
thereby adversely affecting the health of a host (a human).
1 17287359_1 (GHMatters) P114129.AU
Thus, in order to maintain a healthy state for a long time,
it is important to maintain intestinal flora where the
Bifidobacteria are dominant.
[00031
HMOs are contained in breast milk at 10 to 20 g/L, and are a
mixture of 130 or more types of oligosaccharides. Their
structures include 13 different types of core structures to
which fucose and sialic acid are added. The ratio of
fucosylated neutral sugar is high in human milk, and
representatively, include 2'-fucosyl lactose (hereinafter,
also referred to as "2'-FL"), lacto-N-fucopentaose I
(hereinafter, also referred to as "LNFPI"),
lacto-N-difucohexaose I (hereinafter, also referred to as
"LNDFP I"), and lacto-N-tetraose (hereinafter, also referred
to as "LNT") . These four types account for 1/3 to 1/4 of all
human milk oligosaccharides. Oligosaccharides containing
lacto-N-biose are set as type I, and oligosaccharides
containing N-acetyllactosamine (Gal($1-4)G1cNAc) are set as
type II. In human milk, the amount of LNT or LNFP1 of type
Iis higher than that oflacto-N-neotetraose (hereinafter, also
referred to as "LNnT") or lacto-N-fucopentaose III of type II.
The coexistence of type I and type II, and the preference of
type I are distinctive characteristics of human milk
oligosaccharides, which are different from other types of milk
oligosaccharides (see Non-Patent Literatures 1 and 2).
2 17287359_1 (GHMatters) P114129.AU
[00041
HMOs present in breast milk, 2'-FL and LNnT, have a
Bifidobacteria proliferation activity, are known to act as a
growth factor of Bifidobacteria by being selectively used by
Bifidobacteria, and are important in the formation of
Bifidobacteria-dominantintestinal flora. Also, HMOs may be
synthesized, and therefore have industrial applicability (see
Patent Literature 1).
[0005]
Bifidobacteriametabolize some HMOs by using a GNB/LNB pathway
specific to the Bifidobacteria. This pathway is present in
many species often found in babies, but not present in
Bifidobacteria often found in the intestinal tract of adults.
Among the four bacterial species of Bifidobacterium longum
subspecies longum, Bifidobacteriumlongumsubspeciesinfantis,
Bifidobacterium breve, and Bifidobacteriumbifidum, which are
the Bifidobacteria often found in babies, the two bacterial
species of Bifidobacterium longum subspecies infantis and
Bifidobacterium bifidum have been reported as bacterial
species that may use HMOs as a sole carbon source (see
Non-Patent Literature 3).
Citation List
Patent Literature
[00061
3 17287359_1 (GHMatters) P114129.AU
Patent Literature 1: W02014/086373
Non-Patent Literature
[0007]
Non-Patent Literature 1: Japanese Journal of Lactic Acid
Bacteria Vol. 22, No. 1 2011
Non-Patent Literature 2: Milk Science Vol. 56, No. 4 2008
Non-Patent Literature 3: Milk Science Vol. 61, No.2. 2012
Non-Patent Literature 4: THE JOURNAL OF BIOLOGICAL CHEMISTRY
VOL. 286, NO. 40
Summary of Invention
Technical Problem
[0008]
In metabolizing HMOs, as described above, among Bifidobacteria,
there are bacteria having no or low ability to assimilate HMOs,
while there are also bacteria that are able to assimilate HMOs,
such as Bifidobacterium longum subspecies infantis and
Bifidobacterium bifidum. Then, in metabolizing HMOs, for
example, Bifidobacterium longum subspecies infantis
incorporates HMOs into the microbial cells, in use, while
Bifidobacterium bifidum temporarily promotes accumulation of
lactose outside microbial cells by decomposing these
oligosaccharides with an extracellular enzyme (see Non-Patent
Literatures 3 and 4).
[0 0 0 9]
4 17287359 1(GHMtters) P114129.AU
As described above, among bacteria, there are bacteria
having no or low ability to assimilate HMOs. However, even
among such bacteria, there are many bacteria that may be
used as good bacteria. Therefore, even probiotics having
no or low ability to assimilate human milk oligosaccharides
(hereinafter, also referred to as "HMOs") are useful as a
nutritional composition as long as proliferation is possible
in the presence of HMOs.
[0010]
Therefore, the present technology provides a technique that
makes it possible to promote proliferation of probiotics
having a low ability to assimilate human milk
oligosaccharides (HMOs), in the presence of HMOs.
[0011]
The present inventors, etc. observed the fact that
Bifidobacterium bifidum temporarily promote accumulation of
lactose outside microbial cells by decomposing HMOs with an
extracellular enzyme, and then found that proliferation of
probiotics having a low ability to assimilate HMOs is
promoted when probiotics having a low ability to assimilate
HMOs, Bifidobacterium bifidum, and HMOs are co-cultured.
[0012]
That is, the present technology provides a nutritional
5 18618821_1 (GHMatters) P114129.AU composition containing bacteria belonging to Bifidobacterium bifidum, one or more types of probiotics having a low ability to assimilate human milk oligosaccharides, and human milk oligosaccharides.
In the nutritional composition according to the present
technology, as for the human milk oligosaccharides,
2'-fucosyllactose and/or lacto-N-neotetraose may be used.
In the nutritional composition according to the present
technology, as for the probiotics, bacteria of the genus
Bifidobacterium, excluding the bacteria belonging to
Bifidobacterium bifidum, and/or lactobacilli may be used.
In this case, as for the bacteria of the genus Bifidobacterium,
excluding the bacteria belonging to Bifidobacterium bifidum,
at least one bacterium selected from Bifidobacterium longum
subspecies longum, Bifidobacterium adolescentis,
Bifidobacterium pseudocatenulatum, Bifidobacterium animals
subspecies lactis, Bifidobacteriumbreve, and Bifidobacterium
pseudolongum may be used.
Also, as for the lactobacilli, at least one bacterium selected
from Lactobacillus paracasei, Lactobacillus plantarum,
Lactobacillus acidophilus, Lactobacillus rhamnosus, and
Lactobacillus gasseri may be used.
In the nutritional composition according to the present
technology, as for the bacteria belonging to Bifidobacterium
bifidum, at least one bacterium selected from Bifidobacterium
6 17287359_1 (GHMatters) P114129.AU bifidum ATCC29521, Bifidobacterium bifidum (NITE BP-02429),
Bifidobacterium bifidum (NITE BP-1252), Bifidobacterium
bifidum (NITE BP-02431), Bifidobacterium bifidum (NITE BP
02648), Bifidobacterium bifidum (NITE BP-02432), and
Bifidobacterium bifidum (NITE BP-02433) may be used.
[00131
The nutritional composition according to the present
technology may be used for a food/drink composition or
modified milk powder.
The present invention as claimed herein is described in the
following items:
[Item 1]
A nutritional composition containing:
bacteria belonging to Bifidobacterium bifidum;
one or more types of probiotics having a low ability
to assimilate human milk oligosaccharides; and
human milk oligosaccharides,
wherein the bacteria belonging to Bifidobacterium
bifidum are at least one bacterium selected from
Bifidobacterium bifidum ATCC29521, Bifidobacterium bifidum
(NITE BP-2429), Bifidobacterium bifidum (NITE BP-1252),
Bifidobacterium bifidum (NITE BP-2431), Bifidobacterium
bifidum (NITE BP-02648), Bifidobacterium bifidum (NITE BP
02432), and Bifidobacterium bifidum (NITE BP-02433),
7 19016887_1 (GHMatters) P114129.AU wherein the human milk oligosaccharides are 2' fucosyllactose and/or lacto-N-neotetraose, wherein the bacteria belonging to the Bifidobacterium bifidum is Bifidobacterium bifidum that has been cultured in vitro, wherein, when the human milk oligosaccharide is 2' fucosyllactose, the probiotic is at least one bacterium selected from: Bifidobacterium longum subspecies longum,
Bifidobacterium pseudocatenulatum, Bifidobacterium animals
subspecies lactis, Lactobacillus paracasei, Lactobacillus
acidophilus, Lactobacillus rhamnosus and Lactobacillus
gasseri, and
wherein, when the human milk oligosaccharide is lacto
N-neotetraose, the probiotic is at least one bacterium
selected from: Bifidobacterium longum subspecies longum,
Lactobacillus paracasei, Lactobacillus plantarum,
Lactobacillus acidophilus, and Lactobacillus rhamnosus.
[Item 2]
A food/drink composition containing the nutritional
composition described in item 1.
[Item 3]
Modified milk powder containing the nutritional
composition described in item 1.
7A 19016887_1 (GHMatters) P114129.AU
Advantageous Effects of Invention
[0014]
According to the present technology, it is possible to
promote proliferation of probiotics having a low ability to
assimilate human milk oligosaccharides (HMOs), in the
presence of HMOs. The effects described herein are not
necessarily limited, and may be any of the effects described
in the present technology.
Description of Embodiments
[0015]
Hereinafter, a suitable embodiment for carrying out the
present technology will be described. The embodiment to be
described below is described as an example of a
representative embodiment of this disclosure, by which the
scope of the present technology is not to be narrowly
construed. In this specification,
7B 18853677_1 (GHMatters) P114129.AU regarding the numerical range expressed as "lower limit to upper limit, " the upper limit may be "or less" or "less than,
" and the lower limit may be "or more" or "greater than." Also,
in this specification, the percentage is expressed by mass
unless otherwise specified.
[00161
1. Nutritional Composition
A nutritional composition according to the present technology
contains at least the following components: (1) bacteria
belonging to Bifidobacterium bifidum, (2) one or more types
of probiotics having a low ability to assimilate human milk
oligosaccharides, and (3) human milk oligosaccharides.
Hereinafter, each of these components will be described in
detail.
[0017]
(1) Bacteria Belonging to Bifidobacterium bifidum
As for bacteria belonging to Bifidobacteriumbifidum which may
be used in the present technology, one type or two or more types
of known or unknown bacteria belonging to Bifidobacterium
bifidum may be freely selected and used as long as the effect
of the present technology is not impaired. Specifically, for
example, examples may include Bifidobacterium bifidum
ATCC29521, Bifidobacterium bifidum (NITE BP-02429),
Bifidobacterium bifidum (NITE BP-1252), Bifidobacterium
bifidum (NITE BP-02431), Bifidobacterium bifidum (NITE
8 17287359_1 (GHMatters) P114129.AU
BP-02648), Bifidobacterium bifidum (NITE BP-02432),
Bifidobacterium bifidum (NITE BP-02433), Bifidobacterium
bifidum (FERMBP-791), Bifidobacterium bifidum (FERMP-11788),
Bifidobacterium bifidum (FERM P-11791), Bifidobacterium
bifidum (NITE BP-31), Bifidobacterium bifidum G9-1,
Bifidobacterium bifidum BF-1, Bifidobacterium bifidum
Rosell-71, Bifidobacteriumbifidum (NCDO 1453), and the like.
[00181
Bifidobacterium bifidum ATCC29521 may be obtained from
theAmerican Type Culture Collection (address: 12301 Parklawn
Drive, Rockville, Maryland 20852, United States of America)
[0019]
Bifidobacterium bifidum (NITE BP-02429), Bifidobacterium
bifidum (NITE BP-02431), Bifidobacterium bifidum (NITE
BP-02432), and Bifidobacterium bifidum (NITE BP-02433) were
internationally deposited with deposit numbers of NITE
BP-02429, NITEBP-02431, NITEBP-02432, andNITEBP-02433under
the Budapest Treaty at Patent MicroorganismDepository Center,
National Institute of Technology and Evaluation (NITE) (Room
No. 122, 2-5-8 Kazusakamatari Kisarazu Chiba 292-0818), on
February 21, 2017.
[0020]
Bifidobacterium bifidum (NITE BP-1252) was internationally
deposited with a deposit number of NITE BP-1252 under the
Budapest Treaty at Patent Microorganism Depository Center,
9 17287359_1 (GHMatters) P114129.AU
National Institute of Technology and Evaluation (NITE) (Room
No. 122, 2-5-8 Kazusakamatari Kisarazu Chiba 292-0818), on
February 23, 2012.
[0021]
Bifidobacterium bifidum (NITE BP-02648) was deposited with a
deposit number of NITE BP-02648 at Patent Microorganism
Depository Center, National Institute of Technology and
Evaluation (NITE) (RoomNo.122, 2-5-8 KazusakamatariKisarazu
Chiba 292-0818), on February 26, 2018.
[0022]
The strain specified by the above-exemplified strain name is
not limited to the strain itself that has been deposited or
registered in a predetermined institution with the
corresponding strain name (hereinafter, for convenience of
explanation, also referred to as a "deposited strain"), but
also includes a strain substantially equivalent thereto (also
referred to as a "derivative strain" or an "induced strain") .
That is, for example, "Bifidobacterium bifidum ATCC29521" is
not limited to the strain itself that has been deposited in
the depositorywitha depositnumber ofBifidobacteriumbifidum
ATCC29521, but also includes a strain substantially equivalent
thereto.
The strain substantially equivalent to the deposited strain
may be, for example, a derivative strain whose parent strain
is the corresponding deposited strain. The derivative strain
10 17287359_1 (GHMatters) P114129.AU may include a strain bred from the deposited strain or a strain naturally occurring from the deposited strain.
[0023]
As substantially identicalstrains and derivative strains, the
following strains may be mentioned:
(1) a strain determined as an identical strain by an RAPD
(Randomly Amplified Polymorphic DNA) method or a PFGE
(Pulsed-field gel electrophoresis) method (described in
Probiotics in food/Health and nutritional properties and
guidelines for evaluation 85, Page 43)
(2) a strain that has only the genes derived from the
corresponding deposited strain, has no foreign-derived genes,
and has a DNA identity of 95% or more
(3) a strain bred from the corresponding strain (a strain having
the same traits, including genetic engineering alterations,
mutations, and natural mutations)
[0024]
In metabolizing HMOs, such bacteria belonging to
Bifidobacterium bifidum temporarily promote accumulation of
lactose outside of the microbialcells by decomposing HMOs with
an extracellular enzyme. The accumulated lactose may be used
by probiotics having a low ability to assimilate human milk
oligosaccharides, which will be described below, so that the
probiotics having a low ability to assimilate human milk
oligosaccharides may proliferate.
11 17287359_1 (GHMatters) P114129.AU
[00251
(2) Probiotics Having Low Ability to assimilate Human Milk
Oligosaccharides
As for probiotics having a low ability to assimilate human milk
oligosaccharides, which may be used in the present technology,
one type or two or more types of microbial cells may be freely
selected from known or unknown probiotics having a low ability
to assimilate human milk oligosaccharides, as long as the
effect of the present technology is not impaired.
[0026]
In the present invention, "the ability to assimilate human milk
oligosaccharides is low" means that bacteria are not only less
able to utilize sugars, but also less able to metabolize and
proliferate when cultured in a medium containing human milk
oligosaccharides as a carbon source, as compared to when
cultured in a medium containing sugars such as glucose or
lactose, which are generally assimilated by Bifidobacteriaand
lactobacilli (as probiotics), as a carbon source.
For example, the ability to assimilate human milk
oligosaccharides maybe determinedbymeasuring the uptake rate
of human milk oligosaccharides introduced into cells, as an
index, and may be calculated by measuring whether human milk
oligosaccharides are decreasing with time after the start of
culturing. Otherwise, confirmation may be performed by
checking the turbidity or the specific growth rate of
12 17287359_1 (GHMatters) P114129.AU microorganisms in a medium containing only human milk oligosaccharides, as a carbon source.
[00271
The specific growth rate of a microorganisms is defined as an
increase of cells per unit time. The case of "the ability to
assimilate human milk oligosaccharides is low" means that the
specific growth rate is reduced to 80% or less, preferably 70%
or less, more preferably 50% or less, further preferably 10%
or less in a case of human milk oligosaccharides as a carbon
source as compared to that in a mediumhaving glucose or lactose
as a carbon source. Specifically, for example, in the present
technology, as for probiotics having a low ability to
assimilate human milk oligosaccharides, bof the genus
Bifidobacterium, excluding bacteria belonging to
Bifidobacterium bifidum, and/or lactobacilli may be
mentioned.
[00281
In the present invention, probiotic microbial cells having no
assimilability of human milk oligosaccharides may be used.
The probioticmicrobialcells havingno assimilability ofhuman
milk oligosaccharides mean microbial cells which cannot use
human milk oligosaccharides at all, or which are partially
deficient in a metabolicpathway, and do not have a transporter
that introduces metabolized sugar into the body.
[0029]
13 17287359_1 (GHMatters) P114129.AU
More specifically, as for bacteriaof the genus Bifidobacterium,
excluding bacteria belonging to Bifidobacterium bifidum,
Bifidobacterium longum subspecies longum, Bifidobacterium
adolescentis, Bifidobacterium pseudocatenulatum,
Bifidobacterium animals subspecies lactis, Bifidobacterium
breve, Bifidobacterium pseudolongum, and the like may be
mentioned.
[00301
Further specifically, Bifidobacterium longum subspecies
longum ATCC15707, Bifidobacterium longum subspecies longum
BB536, Bifidobacterium adolescentis ATCC15703,
BifidobacteriumpseudocatenulatumATCC27919, Bifidobacterium
animalissubspecieslactisDSM10140, Bifidobacteriumanimalis
subspecies lactis MCC-525, Bifidobacterium breve ATCC15700,
Bifidobacterium pseudolongum ATCC25526, Bifidobacterium
longum (FERM P-10657), Bifidobacterium animals subspecies
lactis Bb-12 DSM1595, Bifidobacterium animals subspecies
lactis GCL2505, Bifidobacterium animals subspecies lactis
CNCMI-2494, Bifidobacterium animals subspecies lactis LAFTI
B94, Bifidobacterium animals subspecies lactis,
Bifidobacterium longum Rosell-175, Bifidobacterium animals
subspecies animals, Bifidobacterium animals subspecies
lactis HN019, and the like may be mentioned.
[00311
Also, as for lactobacilli, Lactobacillus paracasei,
14 17287359_1 (GHMatters) P114129.AU
Lactobacillus plantarum, Lactobacillus acidophilus,
Lactobacillus rhamnosus, Lactobacillus gasseri, and the like
may be mentioned.
[00321
More specifically, Lactobacillus paracasei ATCC25302,
Lactobacillus plantarum ATCC14917, Lactobacillus acidophilus
ATCC4356, Lactobacillus rhamnosus ATCC7469, Lactobacillus
gasseri ATCC33323, Lactobacillus gasseri FERM BP-10953,
Lactobacillus gasseri FERM BP-6999, Lactobacillus gasseri
(NITE BP-224R), Lactobacillus delbrueckii subspecies
bulgaricus (FERMP-17227), Lactobacillus rhamnosusATCC53103,
Lactobacillus reuteri DSM17938, Bacillus coagulans GBI-30,
Lactobacillus acidophilus, Lactobacillus acidophilus LAFTI
L10, Lactobacillus casei LAFTI (registered trademark) L26,
Lactobacillus casei LAFTI (registered trademark) L26,
Lactococcus lactis subspecies lactis Rosell-1058,
Lactobacillus paracasei NCC2461, Lactobacillus johnsonii
NCC533, Lactobacillus rhamnosus Rosell-li, Lactobacillus
acidophilus Rosell-52, Lactobacillus casei Lactobacillus
casei shirota strains, and the like may be mentioned.
[00331
These probiotics having a low ability to assimilate human milk
oligosaccharides may proliferate by using lactose produced
when HMOs are metabolized by the above described bacteria
belonging to Bifidobacterium bifidum. More preferably, it is
15 17287359_1 (GHMatters) P114129.AU desirable that the specificgrowthrate ofprobioticsincreases by culturing together with bacteria belonging to
Bifidobacterium bifidum. For example, it means that the
specific growth rate is increased to 105% or more, preferably
110% or more, more preferably 120% or more in a case of human
milk oligosaccharides as a carbon source as compared to that
in a medium having glucose or lactose as a carbon source.
[00341
In the present technology, "probiotics" mean live bacteria that
exhibit a beneficial action on human health.
[00351
(3) Human Milk Oligosaccharides (HMOs)
HMOs are a generic term for oligosaccharides having three or
more sugars, excludinglactose thatis presentin a smallamount
in breast milk. Mammalian milk has 12 series of core skeletons
such as lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT),
lacto-N-hexaose (LNH), and lacto-N-neohexaose (LNnH). Lewis
a, b, x and a2-3/2-6N-Acetylneuraminic acid are added to these
skeletons, so that there are as many as 100 possible variations
of sugar chains.
[00361
As for HMOs that may be used in the present technology, one
type or two or more types of known or unknown HMOs, which are
oligosaccharides that may be used by bacteria belonging to
Bifidobacterium bifidum, and also may not be used by the
16 17287359_1 (GHMatters) P114129.AU probiotics, may be freely selected and used as long as the effect of the present technology is not impaired.
Specifically, for example, L-fucose, Lacto-N-fucopentaose I,
Lacto-N-fucopentaose II, 2'-Fucosyllactose,
3'-Fucosyllactose, Lacto-N-fucopentaose III,
3'-Fucosyllacto-N-hexaose, Lacto-N-difucohexaose I,
Lacto-difucotetraose, Lacto-difucohexaose I,
Lactodifucohexaose II, Lacto-N-tetraose, Difucosyl
syalyllacto-N-hexaose, Syalyllacto-N-tetraose,
Fucosylsyalyl lacto-N-hexaose I, 3'-Fucosylsialyl
lacto-N-tetraose(Fuc 1, 2 Gal, Fuc 1, 4 Gal NAC),
Lacto-N-neotetraose, 3'-Sialyl-3-fucosyllactose,
Disialomonofucosyllacto-N-neohexaose, Monofucosylmonosialyl
lacto-N-octaose, Sialyllacto-N-fucohexaose II,
Disialyllacto-N-fucopentaose II,
Monofucosyldisialyllacto-N-tetraose, 2'-Sialyllactose,
2'-Sialyllactosamine, 3'-Sialyllactose,
3'-Sialyllactosamine, 6'-Sialyllactose,
6'-Sialyllactosamine, Sialyllacto-N-neotetraose c,
Monosialyllacto-N-hexaose, Disialyllacto-N-hexaose I,
Monosialyllacto-N-neohexaose I,
Monosialyllacto-N-neohexaose II, Disialyllacto-N-neohexaose,
Disialyllacto-N-tetraose, Disialyllacto-N-hexaose II,
Sialyllacto-N-tetraose a, Disialyllacto-N-hexaose I,
Sialyllacto-N-tetraose b, 2'-Fucosyllacto-N-hexaose,
17 17287359_1 (GHMatters) P114129.AU
3'-Fucosyllacto-N-neohexaose,
2'-Fucosyl-N-acetylglucosamine, Lacto-N-fucopentaose V,
Lacto-N-hexaose, Para-lacto-N-hexaose, Lacto-N-neohexaose,
Para-lacto-N-neohexaose, Monofucosyllacto-N-hexaose II,
Isomeric fucosylated lacto-N-hexaose (1), Isomeric
fucosylated lacto-N-hexaose (3), Isomeric fucosylated
lacto-N-hexaose (2), Difucosyl-para-lacto-N-neohexaose,
Difucosyl-para-lacto-N-hexaose, 2'
3'-Difucosyllacto-N-hexaose, Lacto-N-neoocataose,
Para-lacto-N-octanose, Iso-lacto-N-octaose, Lacto-N-octaose,
Monofucosyllacto-neoocataose, Monofucosyllacto-N-ocataose,
Difucosyllacto-N-octaose I, Difucosyllacto-N-octaose II,
Difucosyllacto-N-neoocataose II,
Difucosyllacto-N-neoocataose I, Lacto-N-decaose,
Trifucosyllacto-N-neooctaose, Trifucosyllacto-N-octaose,
Trifucosyl-iso-lacto-N-octaose, Lacto-N-difuco-hexaose II,
Sialyl-lacto-N-tetraose a, Sialyl-lacto-N-tetraose b,
Sialyl-lacto-N-tetraose c, Sialyl-fucosyl-lacto-N-tetraose I,
Sialyl-fucosyl-lacto-N-tetraose II,
Disialyl-lacto-N-tetraose, Lacto-N-neotetraose, etc. and
combinations thereof may be mentioned.
Among these, 2'-fucosyllactose (hereinafter, also referred to
as "2'-FL"), lacto-N-fucopentaose I (hereinafter, also
referred to as "LNFPI"), lacto-N-difucohexaose I (hereinafter,
also referred to as "LNDFP I") and lacto-N-neotetraose
18 17287359_1 (GHMatters) P114129.AU
(hereinafter, also referred to as "LNnT") may be used.
[00371
2. Application of Nutritional Composition according to the
Present Technology
When the nutritional composition of the present invention is
administered, promotion ofproliferation of probiotics acting
on good bacteria may be expected. That is, as the probiotics
proliferate in the intestine, the intestinalfloraisimproved.
The improvement of the intestinal flora makes it possible to
improve or prevent, and treat diseases causedby the intestinal
flora.
As for diseases caused by the intestinal flora, inflammatory
colitis, ulcerative colitis, irritable colitis, a colorectal
cancer, diabetes, arteriosclerosis, and the like may be
mentioned.
Also, when the nutritionalcomposition of the presentinvention
is administered, immune function is enhanced. The enhancement
of the immune function makes it possible to prevent or improve,
and treat allergies such as pollen allergy, asthma, and atopic
dermatitis.
The nutritional composition according to the present
technology may be suitably used in foods/drinks, medicines,
quasi-drugs, feeds, and the like.
[00381
The application of this embodiment may be for a therapeutic
19 17287359_1 (GHMatters) P114129.AU purpose use, or a non-therapeutic purpose use.
The "non-therapeutic purpose" is a concept that does not
include a medical act, that is, an act of treating a human body
by treatment. For example, health promotion, aesthetic
treatment, and the like may be mentioned.
"Improvement" refers to improvement of a disease, a symptom
or a condition; prevention or delay of deterioration of a
disease, a symptom or a condition; or reversal, prevention or
delay of progression of a disease or a symptom.
"Prevention" refers to prevention or delay of the onset of a
disease or a symptom in the application target, or risk
reduction of a disease or a symptom of the application target.
[00391
In each nutritional composition, it is desirable to administer
probiotics at about 103 to about 1012 CFU/g per dosage unit,
but values higher than these are also acceptable. When the
probiotic composition is administered to an adult (that is,
a human over age 16), it is desirable to administer probiotics
at a dose of, preferably, at least 106 CFU/g, more preferably
108 CFU/g. When the probiotic composition is administered to
a child (that is, a human under age 16), it is desirable to
administer probiotics in the range of preferably about 106 to
about 108 CFU/g. CFU indicates a unit for colony formation:
a colony forming unit.
[0 040 ]
20 17287359_1 (GHMatters) P114129.AU
It is desirable that the administration subject of the present
invention is an infant.
Infants include babies and preschoolers, more particularly
include babies, preschoolers, and newborn babies, and more
particularly include babies, preschoolers, newborn babies,
premature babies, pretermbabies, andlowbirthweightinfants.
In the present invention, the species of the infants include
a human unless otherwise specified. The baby refers to a child
in infancy, and the infancy refers to a period during which
milk such as breast milk is a main nutritional source. In the
case ofhumans, the infancyusually corresponds to the age under
1. The preschooler usually refers to a child in a preschool
period. The newborn baby refers to a childin aneonatalperiod,
and the neonatal period means a period soon after the birth.
In the case of humans, the neonatal period usually means within
four weeks after the birth.
[0041]
Also, in the present invention, in addition to human milk
oligosaccharides, prebiotics may also be used together.
Prebiotics are indigestible food ingredients which positively
affect the host and improve the health of the host, by
selectively changing the proliferation and activity of certain
bacteria within the large intestine.
Prebiotics are not particularly limited as long as the effect
of the present invention may be further obtained when they are
21 17287359_1 (GHMatters) P114129.AU ingested together with the present bacteria or a culture thereof, but, for example, lactulose, raffinose, galactooligosaccharides, fructooligosaccharides, soybean oligosaccharides, lacto-fructo oligosaccharides, xylo oligosaccharides, isomalto oligosaccharides, coffee bean manno-oligosaccharides, gluconic acid, polydextrose, inulin, etc. are preferred. Among these, lactulose, raffinose, galactooligosaccharides, and the like are more preferred, and lactulose, raffinose and galactooligosaccharides are further preferred.
[0042]
<Food/Drink>
The nutritional composition according to the present
technology may be added to a conventionally known food/drink
in preparation, or may be mixed with a food/drink raw material
so as to produce a new food/drink.
[0043]
The food/drink using the nutritional composition according to
the present technologymayhave any formsuchas liquids, pastes,
solids, or powder, and not only tablet confectioneries, liquid
foods, etc., but also, for example, flour products, instant
foods, processed agricultural products, processed seafood
products, processed livestock products, milk/dairy products,
oils and fats, basic seasonings, complex seasonings/foods,
frozen foods, confectioneries, drinks, commercially available
22 17287359_1 (GHMatters) P114129.AU products other than these, etc. may be mentioned.
[00441
Examples of the flour product may include bread, macaroni,
spaghetti, noodles, cake mix, fryingpowder, bread crumbs, etc.
Examples of the instant food may include instant noodles, cup
noodles, retort/cooked foods, cooked cans, microwave foods,
instant soup/stew, instantmiso soup/soup, canned soup, freeze
dried foods, other instant foods, etc.
Examples of the processed agricultural product may include
canned agriculturalproducts, canned fruits, jams/marmalades,
pickles, boiled beans, dried agricultural products, cereals
(processed grain products), etc.
Examples of the processed seafood product may include canned
seafoods, fish-flesh hams/sausages, seafood paste foods,
seafood delicacies, tsukudani, etc.
Examples of the processed livestock product may include canned
livestock/pastes, meat hams/sausages, etc.
Examples of the milk/dairy product may include fermented milk,
processed milk, milk drinks, yogurts, lacticbeverages, cheese,
ice creams, modified milk powder, cream, other dairy products,
etc.
Examples of the oil and fat may include butter, margarines,
vegetable oils, etc.
As the basic seasoning, for example, soy sauces, miso, sauces,
processed tomato seasonings, mirins, vinegars, etc. may be
23 17287359_1 (GHMatters) P114129.AU mentioned, and as the complex seasoning/food, cooking mix, curry sauces, spicy soy sauces, dressings, mentsuyu, spices, other complex seasonings, etc. may be mentioned.
Examples of the frozen foodmay include frozen food ingredients,
semi-cooked frozen foods, cooked frozen foods, etc.
Examples of the confectionery may include caramels, candies,
chewing gums, chocolates, cookies, biscuits, cakes, pies,
snacks, crackers, Japanese sweets, rice confectioneries, bean
confectioneries, dessert confectioneries, other
confectioneries, etc.
Examples of the drink may include carbonated drinks, natural
fruit juices, fruit juice drinks, fruit juice-containing soft
drinks, pulp drinks, granule-containing fruit drinks,
vegetable-based drinks, soy milk, soy milk drinks, coffee
drinks, tea drinks, powdered drinks, concentrated drinks,
sports drinks, nutritional drinks, alcohol drinks, other
favorite drinks, etc.
Examples of the other commercially available foods may include
weaning foods, furikake, ochazuke seaweed, etc.
[0045]
The amount of bacteria belonging to Bifidobacterium bifidum
in the food/drink according to the present technology may be
freely set as long as the effect of the present technology is
not impaired. In the present technology, in particular, the
amount of bacteria belonging to Bifidobacteriumbifidum in the
24 17287359_1 (GHMatters) P114129.AU food/drink may be set to 103 to 1012 CFU/mL or g with respect to the final composition of the food/drink.
[0046]
The content of probiotics having a low ability to assimilate
human milk oligosaccharides in the food/drink according to the
present technology may be freely set as long as the effect of
the present technology is not decreased or inhibited. In the
present technology, in particular, the amount of probiotics
having a low ability to assimilate human milk oligosaccharides
in the food/drink may be set to 103 to 1012 CFU/mL or g with
respect to a final composition of the food/drink.
[0047]
The amount of HMOs in the food/drink according to the present
technology may be freely set as long as the effect of the present
technology is not decreased or inhibited. In the present
technology, in particular, the amount of HMOs in the food/drink
maybe set to 0.3% to 5.0% with respect to a final composition
of the food/drink.
[0048]
[Functionality Labeled Food/Drink]
Also, the foods/drinks and the like defined in the present
technology may also be provided or sold as foods/drinks labeled
with a specific application (particularly, a health
application) or a function.
The action of "labeling" includes all actions of informing
25 17287359_1 (GHMatters) P114129.AU consumers of the above application, and all expressions correspond to the action of "labeling" of the present technology regardless of the purpose of a label, the contents of a label, and a target object and a medium of labeling, as long as the above application may be recalled or realized.
[00491
Also, it is desirable that "labeling" is expressed so the
consumers can directly recognize the above application.
Specifically, an action of assignment and delivery of a product
or a product packaging related to a food or a drink (on which
the above application is described), and display and
importation for the assignment or delivery; and an action of
displaying or distributing advertisements, a price list, or
a transaction document related to the product, on which the
above application is described, or providing information
having any of these as its content, on which the above
application is described, by an electromagnetic (the Internet,
etc.) method may be mentioned.
[0050]
Meanwhile, as for the label contents, a label permitted by the
government or the like (for example, a label or the like that
is approved in accordance with various systems specified by
the government, and is applied in the form based on such
approval) is preferred. Also, it is desirable that such label
contents are attached to packaging, containers, catalogs,
26 17287359_1 (GHMatters) P114129.AU pamphlets, advertising materials at a sales site such as a POP, other documents and the like.
[0051]
Also, as for the "label," labels for health foods, functional
foods, foods for patients, enteral nutritional foods, special
purpose foods, health functional foods, foods for specified
health uses, functionality labeled foods, nutritionally
functional foods, quasi-drugs, etc. may also be mentioned.
Among these, particularly, labels approved by the Consumer
Affairs Agency, for example, labels or the like approved by
systems for foods for specified health uses, systems for
functionality labeled foods, and systems similar to these, may
be mentioned. More specifically, labels for foods for
specified health uses, labels for foods for conditional
specified healthuses, labels for functionality labeled foods,
labels to the effect that the structure or function of a body
is affected, labels on disease risk reduction, and the like
may be mentioned. Among these, typical examples include
labels for foods for specified health uses, which are
stipulated in the Enforcement Regulations of Health Promotion
Law (April 30, 2003, Japanese Ministry of Health, Labor, and
Welfare Ordinance No. 86) (particularly, labels for health
applications), labels for functionality labeled foods, which
are stipulated in Food Labeling Act (2013, Law No. 70), and
labels similar thereto.
27 17287359_1 (GHMatters) P114129.AU
[00521
[Modified Milk Powder]
The nutritional composition according to the present
technology may be properly used in foods/drinks, especially,
in modified milk powder. As for the modified milk powder,
modified milk powder for babies, modified milk powder for
follow-up, modified milk powder for low birth weight infants,
modified milk powder for children, modified milk powder for
adults, modifiedmilk powder for elderly, modifiedmilk powder
for allergy, modified milk powder for lactose intolerance,
modified milk powder for inborn errors of metabolism and the
like may be mentioned. Particularly, use for modified milk
powder forbabies, modifiedmilk powder for follow-up, modified
milk powder for low birth weight infants, and modified milk
powder for children is desirable. The modified milk powder
for babies refers to modified milk powder for babies, which
targets 0 to 12-month-old babies, follow-up milk which targets
babies after 6 to 9months and youngpreschoolers (up to 3 years),
modified milk powder for low birth weight infants, which
targetsnewbornbabies (lowbirthweightinfants) weighingless
than 2500 g at birth, various therapeutic milks used for
treatment of children having pathological conditions such as
milk allergy or lactose intolerance, or the like. Also, the
corresponding composition may be applied to health functional
foods or foods for patients. A health functional food system
28 17287359_1 (GHMatters) P114129.AU is provided for not only general foods but also foods in the form of tablets, capsules, etc., as targets, on the premise of domestic and foreign trends, and consistency with a traditional system for foods for specified health uses, and is composed of two types such as types of foods for specified health uses (individual permission type) and nutritionally functional foods (standard reference type).
[00531
When the nutritional composition of the present technology is
administered, Bacteria of the genus Bifidobacterium and/or
lactobacilliproliferatein the intestine of an administration
subject. The proliferation of these probiotics in the
intestine improves immunity, and further improves intestinal
flora. In particular, in infants, when the nutritional
composition of the present invention is administered,
Bifidobacteriainthe intestine are effectively increased, and
the intestinal environment is improved. Also, by
incorporating bad bacteria or opportunistic bacteria together
with Bifidobacteria, immunity may be acquired, and the
intestinal environment such as the entire intestinal bacteria
flora, will be maintained, even when adulthood is reached.
[00541
As a method of producing modified milk powder, one type or two
or more types of known production methods may be freely used
in combination as long as the effect of the present technology
29 17287359_1 (GHMatters) P114129.AU is not decreased or inhibited. Specifically, for example, in the production, raw material milk may be mixed with the nutritional composition according to the present technology and various additives, and subjected to processes such as homogenization, sterilization, concentration, drying, granulation, and sieving.
[00551
Specifically, the modified milk powder of the present
technology may be produced by, for example, the following
method.
[00561
That is, the present technology provides a method of producing
milk powder for enhancing a breast milk component, in which
microbial cell powder, which includes bacteria belonging to
Bifidobacterium bifidum and one or more types of probiotics
havingalow ability to assimilate humanmilk oligosaccharides,
is mixedwithhuman milk oligosaccharides to obtainmilk powder
for enhancing a breast milk component.
Specifically, there is provided a method of producing milk
powder for enhancing a breast milk component, which includes
the following steps (A) to (C):
(A) a step of culturing bacteria belonging to Bifidobacterium
bifidum, and one or more types of probiotics having a low
ability to assimilate human milk oligosaccharides in the
present invention, in a medium containing a milk component to
30 17287359_1 (GHMatters) P114129.AU obtain cultures;
(B) a step of subjecting the cultures to spray-drying and/or
freeze-drying to obtain respective microbialcellpowders; and
(C) a step of mixing the microbial cell powders with human milk
oligosaccharides to obtain modified milk powder.
[00571
Also, the present technology provides a method of producing
modified milk powder, which includes the following step (A) :
(A) a step of mixing human milk oligosaccharides, bacteria
belonging to Bifidobacterium bifidum, one or more types of
probiotics having a low ability to assimilate human milk
oligosaccharides of the invention, and a milk component to
obtain milk powder.
[00581
Also, specifically, the food composition of the present
technology may be, for example, a supplement for babies. The
supplement for babies may be produced by, for example, the
following method.
[00591
That is, the present technology provides a method of producing
a supplement for enhancing a breast milk component, which
includes the following steps (A) and (B):
(A) a step of mixing human milk oligosaccharides, bacteria
belonging to Bifidobacterium bifidum, one or more types of
probiotics having a low ability to assimilate human milk
31 17287359_1 (GHMatters) P114129.AU oligosaccharides, and an excipient to obtain a mixture; and
(B) a step of tableting the mixture.
[00601
In any of the production methods, ingredients other than the
ingredients mentioned in the above steps may be properly used
in combination.
[0061]
A method of adding the nutritional composition according to
the present technology is not particularly limited as long as
the effect of the present technology is not impaired, but, for
example, the nutritional composition according to the present
technology may be added in a liquid state at the time of mixing
as described above, or may be added after formed into powder,
and then powder-mixing may be performed for production.
[0062]
The content of bacteria belonging to Bifidobacterium bifidum
in the modifiedmilk powder according to the present technology
may be freely set as long as the effect of the present technology
is not decreased or inhibited. In the present technology, in
particular, the amount of bacteria belonging to
Bifidobacterium bifidum in the modified milk powder may be set
to 103 to 1012 CFU/g with respect to the final composition of
the modified milk powder.
[00631
The amount of probiotics having a low ability to assimilate
32 17287359_1 (GHMatters) P114129.AU human milk oligosaccharides in the modified milk powder according to the present technology may be freely set as long as the effect of the present technology is not decreased or inhibited. In the present technology, in particular, the amount of probiotics having a low ability to assimilate human milk oligosaccharides in the modified milk powder may be set to 103 to 1012 CFU/g with respect to the final composition of the modified milk powder.
[0064]
The amount of HMOs in the modified milk powder according to
the present technology may be freely set as long as the effect
of the present technology is not decreased or inhibited. In
the present technology, in particular, the amount of HMOs in
the modified milk powder may be set to 0.3% to 5.0% with respect
to the final composition of the modified milk powder.
[0065]
<Medicine, Quasi-Drug>
The nutritional composition according to the present
technology may be added to a conventionally known medicine or
quasi-drug (hereinafter, also referred to as a "medicine,
etc.") in preparation, or may be mixed with raw materials of
the medicine, etc. so as to produce a new medicine, etc.
[0066]
When the nutritional composition according to the present
technology is blended with the medicine, etc., the
33 17287359_1 (GHMatters) P114129.AU corresponding medicine, etc. maybe properly formulated into a desired dosage form according to an administration method such as oral administration or parenteral administration. The dosage form is not particularly limited, but in the case of oral administration, it is possible to formulate, for example, solid preparations such as powder, granules, tablets, troches, and capsules; and liquidpreparations such as solutions, syrups, suspensions, and emulsions. In the case of parenteral administration, it is possible to formulate, for example, suppositories, spray, inhalants, ointment, patches, injections, and the like. In the present technology, formulation of a dosage form for oral administration is preferred.
The formulation may be properly carried out according to a
dosage form by a conventionally known method.
[0067]
In the formulation, the formulation may be performed by
properly blending with a formulation carrier. Also, in
addition to the peptide of the present technology, components
generally used for formulation, such as an excipient, a pH
adjuster, a colorant, and a corrigent, may be used. Also,
components having an effect of preventing, improving and/or
treating diseases or symptoms which are conventionally known
or to be found in the future may be properly used in combination
depending on purposes.
34 17287359_1 (GHMatters) P114129.AU
[00681
As for the formulation carrier, various organic or inorganic
carriers may be used depending on the dosage forms. In the
case of solid preparations, examples of the carrier may include
excipients, binders, disintegrants, lubricants, stabilizers,
corrigents/flavoring agents, etc.
[00691
Examples of the excipient may include sugar derivatives such
as lactose, sucrose, glucose, mannitol, and sorbitol; starch
derivatives such as corn starch, potato starch, a-starch,
dextrin, and carboxymethyl starch; cellulose derivatives such
as crystalline cellulose, hydroxypropyl cellulose,
hydroxypropyl methyl cellulose, carboxymethyl cellulose, and
carboxymethyl cellulose calcium; gum arabic; dextran;
pullulan; silicate derivatives such as light anhydrous silicic
acid, synthetic aluminum silicate, and magnesium
aluminometasilicate; phosphate derivatives such as calcium
phosphate; carbonate derivatives such as calcium carbonate;
and sulfate derivatives such as calcium sulfate.
[0070]
Examples of the binder may include gelatin;
polyvinylpyrrolidone; macrogol and the like, in addition to
the excipients.
[0071]
Examples of the disintegrant may include cellulose derivatives
35 17287359_1 (GHMatters) P114129.AU or chemically modified starch such as sodium croscarmellose, sodium carboxymethyl starch, and cross-linked polyvinylpyrrolidone, in addition to the excipients.
[00721
Examples of the lubricant may include talc; stearic acid;
metallic stearates such as calcium stearate, and magnesium
stearate; colloidal silica; waxes such as bee gum, and
spermaceti; boric acid; glycol; carboxylic acids such as
fumaricacid and adipicacid; carboxylicacid sodiumsalts such
as sodium benzoate; sulfates such as sodium sulfate; leucine;
lauryl sulfates such as sodium lauryl sulfate, and magnesium
lauryl sulfate; silicic acids such as silicic acid anhydride,
and silicic acid hydrate; and starch derivatives.
[0073]
Examples of the stabilizer may include paraoxybenzoic acid
esters such as methylparaben and propylparaben; alcohols such
as chlorobutanol, benzyl alcohol, and phenylethyl alcohol;
benzalkonium chloride; acetic anhydride; and sorbic acid.
[0074]
Examples of the corrigent/flavoring agent may include
sweeteners, acidulants, flavors, etc.
As for the carrier to be usedin the case ofliquidpreparations
for oral administration, a solvent such as water, a
corrigent/flavoring agent, etc. may be mentioned.
[0 075]
36 17287359_1 (GHMatters) P114129.AU
The amount of bacteria belonging to Bifidobacterium bifidum
in the medicine, etc. according to the present technology may
be freely set as long as the effect of the present technology
is not decreased or inhibited. In the present technology, in
particular, the amount of bacteria belonging to
Bifidobacterium bifidum in the medicine, etc. may be set to
103 to 1012 CFU/mL or g with respect to the final composition
of the medicine, etc.
[0076]
The amount of probiotics having a low ability to assimilate
human milk oligosaccharides in the medicine, etc. according
to the present technologymaybe freely set as long as the effect
of the present technology is not decreased or inhibited. In
the present technology, in particular, the content of
probiotics having a low ability to assimilate human milk
oligosaccharides in the medicine, etc. may be set to 103 to 1012
CFU/mL or g with respect to the final composition of the
medicine, etc.
[0077]
The amount of HMOs in the medicine, etc. according to the
present technology may be freely set as long as the effect of
the present technology is not impaired. In the present
technology, in particular, the amount of HMOs in the medicine,
etc. may be set to 0.3% to 5.0% with respect to a final
composition of the medicine, etc.
37 17287359_1 (GHMatters) P114129.AU
[00781
<Feed>
The nutritional composition according to the present
technology may be added to a conventionally known feed in
preparation, or may be mixed with raw materials of the feed
so as to produce a new feed.
[0079]
When the nutritional composition according to the present
technology is blended with the feed, examples of raw materials
of the feed may include cereals such as corn, wheat, barley,
and rye; brans such as wheat bran, barley bran, rice bran, and
defatted rice bran; by-product feeds such as corn gluten meal
and corn germmeal; animal feeds such as skim milk powder, whey,
fish meal, and bone meal; yeasts such as beer yeast; mineral
feeds such as calcium phosphate, and calcium carbonate; oils
and fats; amino acids; and sugars. Also, examples of the form
of the feed may include pet feeds (pet foods and the like),
livestock feeds, fish feeds, and the like.
[0080]
The amount of bacteria belonging to Bifidobacterium bifidum
in the feed according to the present technology may be freely
set according to the body weight, etc. as long as the effect
of the present technology is not decreased or inhibited. In
the present technology, in particular, the amount of bacteria
belonging to Bifidobacterium bifidum in the feed may be set
38 17287359_1 (GHMatters) P114129.AU to 103 to 1012 CFU/mL or g with respect to the final composition of the feed.
[0081]
The amount of probiotics having a low ability to assimilate
human milk oligosaccharides in the feed according to the
present technology may be freely set according to the body
weight, etc. as long as the effect of the present technology
is not decreased or inhibited. In the present technology, in
particular, the amount of probiotics having a low ability to
assimilate human milk oligosaccharides in the feed may be set
to 103 to 1012 CFU/mL or g with respect to the final composition
of the feed.
[0082]
The amount of HMOs in the feed according to the present
technology may be freely set according to the body weight, etc.
as long as the effect of the present technology is not decreased
or inhibited. In the present technology, in particular, the
content of HMOs in the feed may be set to 0.3% to 5.0% with
respect to the final composition of the feed.
[0083]
The present technology may also employ the following
configurations.
[1] A nutritional composition containing:
bacteria belonging to Bifidobacterium bifidum;
one or more types of probiotics having a low ability to
39 17287359_1 (GHMatters) P114129.AU assimilate human milk oligosaccharides; and human milk oligosaccharides.
[2] A food/drink composition containing:
bacteria belonging to Bifidobacterium bifidum;
one or more types of probiotics having a low ability to
assimilate human milk oligosaccharides; and
human milk oligosaccharides.
[3] Modified milk powder containing:
bacteria belonging to Bifidobacterium bifidum;
one or more types of probiotics having a low ability to
assimilate human milk oligosaccharides; and
human milk oligosaccharides.
[4] A medicine or quasi-drug composition containing:
bacteria belonging to Bifidobacterium bifidum;
one or more types of probiotics having a low ability to
assimilate human milk oligosaccharides; and
human milk oligosaccharides.
[5] A feed composition containing:
bacteria belonging to Bifidobacterium bifidum;
one or more types of probiotics having a low ability to
assimilate human milk oligosaccharides; and
human milk oligosaccharides.
[6] Use of bacteria belonging to Bifidobacterium bifidum, one
or more types of probiotics having a low ability to assimilate
human milk oligosaccharides, and human milk oligosaccharides,
40 17287359_1 (GHMatters) P114129.AU in a nutritional composition.
[7] Use of bacteria belonging to Bifidobacterium bifidum, one
or more types of probiotics having a low ability to assimilate
human milk oligosaccharides, and human milk oligosaccharides,
in a food/drink composition.
[8] Use of bacteria belonging to Bifidobacterium bifidum, one
or more types of probiotics having a low ability to assimilate
human milk oligosaccharides, and human milk oligosaccharides,
in modified milk powder.
[9] Use of bacteria belonging to Bifidobacterium bifidum, one
or more types of probiotics having a low ability to assimilate
human milk oligosaccharides, and human milk oligosaccharides,
in a medicine or quasi-drug composition.
[10] Use of bacteria belonging to Bifidobacteriumbifidum, one
or more types of probiotics having a low ability to assimilate
human milk oligosaccharides, and human milk oligosaccharides,
in a feed composition.
[11] Use of bacteria belonging to Bifidobacteriumbifidum, one
or more types of probiotics having a low ability to assimilate
human milk oligosaccharides, and human milk oligosaccharides,
in producing a nutritional composition.
[12] Use of bacteria belonging to Bifidobacteriumbifidum, one
or more types of probiotics having a low ability to assimilate
human milk oligosaccharides, and human milk oligosaccharides,
in producing a food/drink composition.
41 17287359_1 (GHMatters) P114129.AU
[13] Use of bacteria belonging to Bifidobacterium bifidum, one
or more types of probiotics having a low ability to assimilate
human milk oligosaccharides, and human milk oligosaccharides,
in producing modified milk powder.
[14] Use of bacteria belonging to Bifidobacteriumbifidum, one
or more types of probiotics having a low ability to assimilate
human milk oligosaccharides, and human milk oligosaccharides,
in producing a medicine or quasi-drug composition.
[15] Use of bacteria belonging to Bifidobacteriumbifidum, one
or more types of probiotics having a low ability to assimilate
human milk oligosaccharides, and human milk oligosaccharides,
in producing a feed composition.
[16] In [1] to [15],
the human milk oligosaccharides are 2'-fucosyllactose and/or
lacto-N-neotetraose.
[17] In [1] to [16],
the probiotics are bacteria of the genus Bifidobacterium
(excluding the bacteria belonging to Bifidobacterium bifidum)
and/or lactobacilli.
[18] In [17],
the bacteria of the genus Bifidobacterium (excluding the
bacteria belonging to Bifidobacterium bifidum) are at least
one bacterium selected from Bifidobacterium longum subspecies
longum, Bifidobacterium adolescentis, Bifidobacterium
pseudocatenulatum, Bifidobacteriurn anirnalis subspecies
42 17287359_1 (GHMatters) P114129.AU lactis, Bifidobacterium breve, and Bifidobacterium pseudolongum.
[19] In [17],
the lactobacilli are at least one bacterium selected from
Lactobacillus paracasei, Lactobacillus plantarum,
Lactobacillus acidophilus, Lactobacillus rhamnosus, and
Lactobacillus gasseri.
[20] In [1] to [19],
the bacteria belonging to Bifidobacteriumbifidum are at least
one bacterium selected from Bifidobacterium bifidum ATCC29521,
Bifidobacterium bifidum (NITE BP-02429), Bifidobacterium
bifidum (NITE BP-1252), Bifidobacterium bifidum (NITE
BP-02431), Bifidobacterium bifidum (NITE BP-02648),
Bifidobacterium bifidum (NITE BP-02432), and Bifidobacterium
bifidum (NITE BP-02433).
[21] Amethod ofproducingmodified milk powder, which includes
the following steps (A) to (C).
(A) a step of culturing bacteria belonging to Bifidobacterium
bifidum, and one or more types of probiotics having a low
ability to assimilate human milk oligosaccharides, in a medium
containing a milk component to obtain cultures;
(B) a step of subjecting the cultures to spray-drying and/or
freeze-drying to obtain respective microbialcellpowders; and
(C) a step of mixing the microbial cell powders with human milk
oligosaccharides to obtain modified milk powder.
43 17287359_1 (GHMatters) P114129.AU
[22] Amethod ofproducingmodified milk powder, which includes
the following step (A):
(A) a step of mixing human milk oligosaccharides, bacteria
belonging to Bifidobacterium bifidum, one or more types of
probiotics having a low ability to assimilate human milk
oligosaccharides, and a milk component to obtain milk powder.
[23] A method of producing a supplement for enhancing a breast
milk component, which includes the following steps (A) and (B)
. (A) a step of mixing human milk oligosaccharides, bacteria
belonging to Bifidobacterium bifidum, one or more types of
probiotics having a low ability to assimilate human milk
oligosaccharides, and an excipient to obtain a mixture; and
a step of tableting the mixture.
[24] In the method described in [21] or [22], the milk component
is a milk protein.
[25] In the method described in [24], the milk protein is at
least one component selected from the group including whey,
whey hydrolysate, and casein.
[26] A method of producing a supplement, which includes the
following steps (A) and (B):
(A) a step of mixing bacteria of the genus Bifidobacterium,
and an excipient to obtain a mixture; and
a step of tableting the mixture.
[27] In [21] to [26],
the human milk oligosaccharides are 2'-fucosyllactose and/or
44 17287359 1(GHMtters) P114129.AU lacto-N-neotetraose.
[28] In [21] to [27],
the probiotics are Bacteria of the genus Bifidobacterium
(excluding the bacteria belonging to Bifidobacterium bifidum)
and/or lactobacilli.
[29] In [28],
the bacteria of the genus Bifidobacterium (excluding the
bacteria belonging to Bifidobacterium bifidum) are at least
one bacterium selected from Bifidobacterium longum subspecies
longum, Bifidobacterium adolescentis, Bifidobacterium
pseudocatenulatum, Bifidobacterium animalis subspecies
lactis, Bifidobacterium breve, and Bifidobacterium
pseudolongum.
[30] In [28],
the lactobacilli are at least one bacterium selected from
Lactobacillus paracasei, Lactobacillus plantarum,
Lactobacillus acidophilus, Lactobacillus rhamnosus, and
Lactobacillus gasseri.
[31] In [21] to [30], in the nutritional composition described
in any one of Claims 1 to 5,
the bacteria belonging to Bifidobacteriumbifidum are at least
one bacterium selected from Bifidobacterium bifidum ATCC29521,
Bifidobacterium bifidum (NITE BP-2429), Bifidobacterium
bifidum (NITE BP-1252), Bifidobacterium bifidum (NITE
BP-2431), Bifidobacterium bifidum (NITE BP-02648),
45 17287359_1 (GHMatters) P114129.AU
Bifidobacteriumbifidum (NITE BP-02432), and Bifidobacterium
bifidum (NITE BP-02433).
Examples
[00841
Hereinafter, the present technology will be described on the
basis of Examples in more detail. Examples described below
are described as examples of a representative Example of the
present technology, by which the scope of the present
technology is not to be narrowly construed.
[0085]
<Experimental Method>
(1) Preparation of Sugar Solution
A10% solution of glucose (manufactured by Nakarai Tesque Inc.),
and 2'-FL ((> 95%) Funakoshi, available from sigma), and LNnT
(manufactured by ELICITYL) as examples of HMOs was prepared
with ion exchanged water, and was subjected to sterile
filtration using Millex-GV (SLGV033RS).
[0086]
(2) Bacterial Solution Preparation of Bacteria of the Genus
Bifidobacterium and Lactobacilli
Lactobacilli MRS Broth (BD) (0.05% L-Cysteine Hydrochloride
for Bacteria of the genus Bifidobacterium) was added to strains
of Table 1 below (bacteria belonging to Bifidobacterium
bifidum) and Table 2 below. After a sterilization treatment,
46 17287359_1 (GHMatters) P114129.AU
3% of the bacterial solution was inoculated, and anaerobically
cultured (pre-pre cultured) at 370C for 16 h.
The following day, 1% glucose was added to a modified BL agar
medium ("Studies on Bifidobacteria" by Tomotari Mitsuoka, p.
306, Japan Bifidus Foundation, issued in 1994) noted in Table
3, for preparation. After a sterilization treatment, 3% of
the pre-pre cultured bacterial solution was inoculated, and
anaerobically pre-cultured (pre-cultured) at 37°C for 12 h.
The pre-cultured bacterial solution was collected and washed
once with a sterile physiological saline solution. Then,
mass-up was performed to an equal volume with the modified BL
agar medium ("Studies on Bifidobacteria" by Tomotari Mitsuoka,
p. 306, Japan Bifidus Foundation, issuedin1994) notedin Table
3 to prepare a suspended bacterial solution.
[0087]
(3) Preparation of Medium for Analysis of the Number of
Proliferating Bacteria in Bacteria of the genus
Bifidobacterium and Lactobacilli
The modified BL agar medium ("Studies on Bifidobacteria" by
Tomotari Mitsuoka, p. 306, Japan Bifidus Foundation, issued
in 1994) noted in Table 3 was prepared at a capacity of 60%,
and was subjected to an autoclave sterilization treatment.
Then, 40% of each sugar solution prepared in (1) was
aseptically added so as to prepare a medium with a sugar
concentration of 4%.
47 17287359_1 (GHMatters) P114129.AU
[00881
(4) Single Bacterial Culturing of Strains of Table 1 below and
Table 2 below
The mediumprepared in the above (3) was dispensed at a capacity
that is half the capacity for culturing in a 96-well plate,
and the bacterial solution prepared in the above (2) was
dispensed at a remaining half capacity such that the bacterial
solution had 6%. Then, a final sugar concentration was set
to 2%. The plate into which the bacterial solution was
dispensed was anaerobically cultured at 370C for 8 h.
[00891
(5) Mix-culturing of bacteria Belonging to Bifidobacterium
bifidum and Probiotics Having Low Ability to assimilate Human
Milk Oligosaccharides
The medium prepared in the above (3) was dispensed at a capacity
that is half the capacity for aseptic culturing in a 96-well
plate, and each of the bacterial solutions of strains of
bacteria belonging to Bifidobacterium bifidum in Table 1 and
probiotics having a low ability to assimilate human milk
oligosaccharides in Table 2, which was prepared in the above
(2), was dispensed at a remaining quarter capacity into the
medium prepared in the above (3) such that the bacterial
solution had 12%. Then, a final sugar concentration was set
to 2%. The plate into which the bacterial solution was
dispensed was anaerobically cultured at 370C for 8 h.
48 17287359_1 (GHMatters) P114129.AU
[00901
(6) Method of Analyzing the Number of Proliferating Bacteria
in Bacteria of the genus Bifidobacterium and Lactobacilli
OD650 was measured from each well in the plates in the above
(4) and the above (5) before (0 h) culturing and after (8 h)
culturing, by MICROPLATE READER SH-9000 (manufactured by
CORONA ELECTRIC). After turbidity measurement, microbial
cells were collected, and DNAextraction was performed. After
the DNA extraction, the number of proliferating bacteria of
Bifidobacteria/lactobacilli was analyzed by using the
bacterial species-specific primer noted in Table 4, by a
quantitative PCR method (see "Structural analysis of
intestinal flora"-analysis of bacterial flora in human feces
using quantitative PCR- by Kouichi Watanabe,
Enterobacteriological journal, 20:35-42, 2006).
[0091]
[Table 1]
Table 1: Bacteria belonging to Bifidobacterium bifidum Strain Bifidobacterium bifidum ATCC29521T Bifidobacterium bifidum NITE BP-02429 Bifidobacterium bifidum NITE BP -1252 Bifidobacterium bifidum NITE BP-02431 Bifidobacterium bifidum NITE P-02648 Bifidobacterium bifidum NITE BP-02432 Bifidobacterium bifidum NITE BP-02433 T:type strain
[0092]
49 17287359_1 (GHMatters) P114129.AU
[Table 2]
Table 2: Bacteria used in Examples except for Bifidobacteriumbifidum Strain Bifidobacterium iongum subsp iongum ATCC15707T Bifidobacterium iongum subsp iongum BB536 Bifidobacterium iongum subsp infantis ATCC 15 6 9 7T
Bifidobacteriumbreve ATCC 15 7 0 0 T
Bifidobacteriumadolescentis ATCC15703T Bifidobacteriumpeseudocatenulatum ATCC27919T BifidobacteriumpseudoIongum ATCC25526T Bifidobacteriumanimalis subsp lactis DSM10140T Bifidobacteriumanimalis subsp lactis MCC-525 Lactobacillusparacasei ATCC25302T Lactobacillusplantarum ATCC 14 9 17T
Lactobacillusacidophhilus ATCC4356T Lactobacillusrhamnosus ATCC7469T Lactobacillusgasseri ATCC33323T T:type strain
[0093]
[Table 3]
50 17287359_1 (GHMatters) P114129.AU
Table 3: Modified BL agar medium 'LAB-LEMCO'POWDER(Oxoid) 2.4 g Bacto Proteose Peptone No. 3(BD) 10.0 g BBL Trypticase Peptone(BD) 5.0 g BBL Phytone Peptone(BD) 3.0 g Bacto Yeast extract(BD) 5.0 g Liver extract 150.0 mL Soluble starch(MERCK) 0.5 g Solution A 10.0 mL Solution B 5.0 mL Tween 80 (KANTO CHEMICAL) 1.0 g L-Cysteine Hydrochloride (KANTO CHEMICAL) 0.5 g pH7.2
Production method of liver extract: 10 g of Bacto Liver (BD), in 170 mL of purified water, is leached for about 1 h in a hot bath of 50 to 60°C, heated at100°C for several minutes, and filtered through filter paper after pH is corrected to 7.2.
Preparation method of solution A: 25 g of KH 2 PO 4 and 25 g of K 2HPO 4 are dissolved in 250 mL of purified water
2 0, 0.5 g of FeSO 4 Preparation method of solution B: 10 g ofMgSO 4 -7H 2 0, 0.5 g of NaCl, -7H and 0.337 g of MnSO 4 are dissolved in 250 mL of purified water
[00941
[Table 4]
51 17287359_1 (GHMatters) P114129.AU
- ~
~) ~) ~) ~) ~)
~ - - -~ ~
~H~HH<<~~DUU U < U
H H ~UU U <QQH~~~<Q ~)
0 0
~ - ~ ~ Cl ~ Cl ~j
- ~ o ~ 0 0
0 * 0
o -~ ~ ~) ~;:r.~ ~ ~ -~ ~
~ ~.- ~
~- ~ ~ ~ ~ ~
[00951
52 172873591(GHM~tte~s)P114129.AU
<Measurement Results>
(1) Availability of HMOs by Bacteria belonging to
Bifidobacterium bifidum
Regarding bacteria belonging to Bifidobacterium bifidum in
Table 1, each strain was cultured as a single bacteriumby 2'-FL
or LNnT (HMOs) as a sugar source, and OD650 was measured. The
results are noted in Table 5 below. Table 5 illustrates OD650
increase values after seven strains of the bacteria belonging
to Bifidobacterium bifidum in Table 1 were cultured for 8 h
by 2'-FL or LNnT as a sugar source.
[00961
[Table 5]
Table 5: OD650 increase value ATCC NITE NITE NITE NITE NITE NITE 29521 BP-02429 BP-1252 BP-02431 P-02648 BP-02432 BP-02433 2'-FL 0.060 0.167 0.225 0.037 0.470 0.430 0.431 LNnT 0.008 0.608 0.373 0.535 0.719 0.522 0.370 T:type strain
[00971
As illustrated in Table 5, all the bacteria belonging to
Bifidobacterium bifidum in Table 1 had assimilability of 2'-FL
or LNnT (HMOs).
[00981
(2) Availability of HMOs by Probiotics having Low Ability to
assimilate Human Milk Oligosaccharides
Regarding strains in Table 2, each strain was cultured as a
53 17287359_1 (GHMatters) P114129.AU single bacterium by 2'-FL or LNnT (HMOs) as a sugar source, and OD650 was measured. The results for 2'-FL are noted in
Table 6 below, and the results for LNnT are noted in Table 7
below.
[00991
[Table 6]
t8~
Q Cl
Qenc (-I
54 17287359_1 (GHMatters) P114129.AU
[0100]
[Table 7]
Table 7: OD650 increase value NITE ATCC BP-02429 BB536 MCC-525 T
(PC) 25302 LNnT 0.608 0.053 0.096 0.052 P C: positive control Ttype strain
[0101]
As illustrated in Tables 6 and 7, among strains in Table 2,
all the strains except for Bifidobacterium infantis which is
considered to have availability, hadno assimilabilityin 2'-FL
or LNnT (HMOs), or a low assimilability.
[0102]
(3) Mix-Culturing of Bacteria belonging to Bifidobacterium
bifidum and Probiotics having Low Ability to assimilate Human
Milk Oligosaccharides by HMOs as Sugar Source
[Bifidobacterium bifidum (NITE BP-02429) + Probiotics Having
Low Ability to assimilate Human Milk Oligosaccharides]
It was decided to culture Bifidobacterium bifidum (NITE
BP-02429) in which availability of 2'-FL or LNnT was found as
described above, in combination with strains in Table 2, which
cannot use 2'-FL or LNnT. Bifidobacterium bifidum (NITE
BP-02429) and the strains in Table 2, which cannot use 2'-FL
or LNnT, were mix-cultured by 2'-FL or LNnT as a sugar source,
55 17287359_1 (GHMatters) P114129.AU and after culturing for 8 h, the proliferation rate of the number of bacteria of each strain (proliferation rate= the number of bacteria after mix-culturing for 8 h / the number of bacteria after single bacterial culturing) was measured by a quantitative PCR method using each strain-specific oligonucleotide primer notedin Table 4, for the strain in Table
2. The results are noted in Table 8.
[01031
[Table 8]
56 17287359_1 (GHMatters) P114129.AU
CaC
biium (NITEBP-0429)
Ca
172739_ (G atr) P1429A 0 H0
[01041
[Bacteria Belonging to Bifidobacterium Bifidumn +
57 17287359 1(GHMtters) P114129.AU
Bifidobacterium longum subspecies longum BB536]
Seven strains of Bifidobacterium bifidum in Table 1 and
Bifidobacterium longum subspecies longum BB536 which cannot
use 2'-FL or LNnT were mix-cultured by 2'-FL or LNnT as a sugar
source. The number of bacteria of Bifidobacterium longum
subspecies longum BB536 after culturing for 8 h, and the number
of bacteria after 8 h of single bacterial culturing were
measured by a quantitative PCR method using Bifidobacterium
longum subspecies longum-specific oligo nucleotide primer
noted in Table 4, and proliferation rates (proliferation
rate=the number of mix-cultured bacteria after 8 h / the number
ofbacteriain 8 hofsingle bacterialculturing) were compared.
The results are noted in Table 9.
[0106]
[Table 9]
Table 9: Proliferation rate ATCC NITE NITE NITE NITE NITE NITE 29521 BP-02429 BP-1252 BP-02431 P-02648 BP-02432 BP-02433 2'-FL 1.35 4.13 3.17 6.33 9.97 3.16 5.90 LNnT 1.07 3.57 1.64 3.02 4.95 1.51 3.08 T:type strain
[0107]
As illustrated in Table 9, it was found that Bifidobacterium
longum subspecies longum BB536 having no assimilability of
2'-FL proliferates through mix-culturing with bacteria
belonging to Bifidobacterium bifidum. Also, it was found that
58 17287359_1 (GHMatters) P114129.AU
Bifidobacterium longum subspecies longum BB536 having no
assimilability ofLNnT proliferates throughmix culturing with
bacteria belonging to Bifidobacterium bifidum.
[0108]
[Bacteria Belonging to Bifidobacterium bifidum
+ Bifidobacterium animalis subspecies lactis MCC-525]
Seven strains of Bifidobacterium bifidum in Table 1 and
Bifidobacterium animalis subspecies lactis MCC-525 which
cannot use 2'-FL or LNnT were mix-cultured by 2'-FL or LNnT
as a sugar source. The number of bacteria of Bifidobacterium
animalis subspecies lactis MCC-525 after culturing for 8 h,
and the number of bacteria after 8 h of single bacterial
culturing were measured by a quantitative PCR method using
Bifidobacterium animalis subspecies lactis-specific oligo
nucleotide primer in Table 4, and proliferation rates
(proliferation rate= the number ofmix-culturedbacteria after
8 h / the number ofbacteriain 8 hofsingle bacterialculturing)
were compared. The results are noted in Table 10.
[0109]
[Table 10]
Table 10: Proliferation rate ATCC NITE NITE NITE NITE NITE NITE 29521 BP-02429 BP-1252 BP-02431 P-02648 BP-02432 BP-02433 2'-FL 2.01 4.85 2.06 3.82 4.35 2.45 3.67 LNnT 3.87 4.17 19.98 3.58 6.03 4.21 2.39 T:type strain
59 17287359_1 (GHMatters) P114129.AU
[0110]
In all combinations with Bifidobacteriurn bifidurn, it was found
that when Bifidobacteriurnanirnalis subspecies lactis MCC-525
was cultured in combination with seven strains of
Bifidobacterium bifidum by 2'-FL or LNnT as a sugar source,
the number of proliferating bacteria was promoted as compared
to that in a case where Bifidobacterium animals subspecies
lactisMCC-525 was cultured as a single bacterium. Among them,
in the combination with Bifidobacteriumbifidum (NITE BP-1252),
the proliferation promotion effect of the number of bacteria
was significantly higher as compared to when LNnT was set as
a sugar source.
[0111]
[Bacteria Belonging to Bifidobacterium bifidum
+ Lactobacillus gasseri ATCC33323]
Seven strains of Bifidobacterium bifidum in Table 1 and
Lactobacillus gasseri ATCC 3 33 2 3T which cannot use 2'-FL were
mix-culturedby2'-FL as a sugar source. The number ofbacteria
of Lactobacillus gasseri ATCC 33 3 2 3 T after 8 h, and the number
of bacteria after 8 h of single bacterial culturing were
measured by a quantitative PCR method using Lactobacillus
gasseri specific-oligo nucleotide primer in Table 4, and
proliferation rates (proliferation rate= the number of
mix-cultured bacteria after 8 h / the number of bacteria in
8 h of single bacterial culturing) were compared. The results
60 17287359_1 (GHMatters) P114129.AU are noted in Table 11.
[0112]
[Table 11]
Table 11: proliferation rate ATCC NITE NITE NITE NITE NITE NITE 2 9 5 2 1T BP-02429 BP-1252 BP-02431 P-02648 BP-02432 BP-02433 2'-FL 1.45 2.33 3.99 4.42 2.88 3.99 3.64 T-type strain
[0113]
In all combinations, it was found that when Lactobacillus
gasseri ATCC33323T was cultured in combination with seven
strains of Bifidobacteriumbifidumby 2'-FL as a sugar source,
the number of proliferating bacteria was promoted as compared
to the number of bacteria in a case where Lactobacillus gasseri
ATCC33323T was cultured as a single bacterium. Among seven
strains of Bifidobacterium bifidum, the proliferation effect
was higher in six strains other than Bifidobacterium bifidum
ATCC29521T.
[0114]
[Bacteria belonging to Bifidobacterium bifidum +
Lactobacillus paracasei ATCC 25302T]
Seven strains of Bifidobacterium bifidum in Table 1 and
Lactobacillus paracasei ATCC 2 5 3 02T which cannot use 2'-FL or
LNnT were mix-cultured by 2'-FL or LNnT as a sugar source.
After 8 h, the proliferation rate of the number of bacteria
of Lactobacillus paracasei ATCC 25302T, with respect to the
61 17287359_1 (GHMatters) P114129.AU number of bacteria after single bacterial culturing
(proliferation rate= the number ofmix-culturedbacteria after
8 h/ the number ofbacteriain 8 h of single bacterial culturing)
was measured by a quantitative PCR method. The results are
noted in Table 12.
[0115]
[Table 12]
Table 12: Proliferation ratio ATCC NITE NITE NITE NITE NITE NITE 2 9 5 2 1T BP-02429 BP-1252 BP-02431 P-02648 BP-02432 BP-02433 2'-FL 2.28 4.21 1.98 3.25 4.23 2.01 3.23 LNnT 4.26 8.04 9.18 10.10 14.78 8.76 8.85 T:type strain
[0116]
In all combinations, it was found that when Lactobacillus
paracasei ATCC 25302T was cultured in combination with seven
strains of Bifidobacterium bifidum by 2'-FL or LNnT as a sugar
source, the number of proliferating bacteria was promoted as
compared to that in a case where Lactobacillus paracaseiATCC
3 02T was cultured as a single bacterium.
[0117]
[Production Example]
Hereinafter, Production Examples of a nutritional composition,
a medicine composition, and a food composition will be
described.
[0118]
62 17287359_1 (GHMatters) P114129.AU
<Production Example 1>
Bifidobacterium bifidum is added to 3 mL of MRS liquid medium,
and is anaerobically cultured at 370C for 16 h. Then, the
culture solution is concentrated, and freeze-dried to obtain
freeze-dried powder of bacteria (bacterial powder). The
bacterial powder containing Bifidobacterium bifidum is
uniformly mixed with probiotics/human milk oligosaccharides
(available from Funakoshi)/whey protein concentrate (WPC),
and prebiotics (lactulose, raffinose and
galactooligosaccharides) to obtain a composition. 20 g of the
corresponding composition is dissolved in 200 g of water so
as to obtain a nutritional composition.
[0119]
<Production Example 2>
Bifidobacterium bifidum is added to 3 mL of MRS liquid medium,
and is anaerobically cultured at 370C for 16 h. Then, the
culture solution is concentrated, and freeze-dried to obtain
freeze-dried powder of bacteria (bacterial powder). The
bacterialpowder is uniformly mixedwithprobiotics/human milk
oligosaccharides (available from Funakoshi)/dry powder of a
milk protein concentrate (MPC480, manufactured by Fonterra,
protein content 80% bymass, casein protein: whey protein=about
8:2), and prebiotics (lactulose, raffinose and
galactooligosaccharides). 20 g of the corresponding
composition is dissolved in 200 g of water to obtain a
63 17287359_1 (GHMatters) P114129.AU nutritional composition.
[01201
<Production Example 3>
Bifidobacterium bifidum is added to 3 mL of MRS liquid medium,
and is anaerobically cultured at 370C for 16 h. Then, the
culture solution is concentrated, and freeze-dried to obtain
freeze-driedpowder ofbacteria (bacterialpowder). Then, the
bacterial powder containing Bifidobacterium bifidum,
probiotics/human milk oligosaccharides (available from
Funakoshi)/prebiotics (lactulose, raffinose and
galactooligosaccharides), and crystalline cellulose are put
into a stirring granulator and mixed. Then, granulation is
performed with addition of purified water, and the granulated
product is dried. Then, the granulated product (a medicine
composition) that contains an extract component of bacteria
and pre/probiotics, and contains an excipient is obtained.
[0121]
<Production Example 4>
A method of producing fermented milk to which Bifidobacterium
bifidum is added will be described below.
First, amilk rawmaterialismixedwithwater, other components,
or the like as necessary, and is preferably subjected to a
homogenization treatment and a heat-sterilization treatment.
The homogenization treatment and the heat-sterilization
treatment may be performed by conventional methods. A
64 17287359_1 (GHMatters) P114129.AU lactobacilli starter is added (inoculated) to the sterilized milk preparation obtained through heat-sterilization, and fermentation is performed while a predetermined fermentation temperature is maintained, so that a fermented product is obtained. By the fermentation, curds are formed.
[0122]
As for the lactobacilli starter, for example, lactobacilli
generally used for yogurt production, such as Lactobacillus
bulgaricus, Lactococcus lactis, and Streptococcus
thermophilus may be used. When pH reaches a target value, the
formed curds are crushed by stirring, and cooled to 100C or
less to obtain a fermented product. By cooling to 10°C or less,
the activation of lactobacilli may be reduced and the
production of acid may be suppressed.
[0123]
Then, the fermented product obtained through the fermentation
process is subjected to heat treatment to obtain a heated
fermented product (a heat-treated fermented product). By
properly heating the fermented product, it is possible to
suppress acid frombeingproduced by lactobacilliinthe heated
fermented product. Accordingly, during the subsequent
production process and/or during storage of concentrated
fermented milk containing Bifidobacteria, reduction of pH may
be suppressed, and as a result, the viability of Bifidobacteria
may be improved.
65 17287359_1 (GHMatters) P114129.AU
[01241
Next, Bifidobacteriumbifidum and human milk oligosaccharides
(available from Funakoshi) are added to the heated fermented
product obtained through the heat treatment process. The
addition amount of Bifidobacterium breve MCC1274 (FERM
BP-11175) is preferably 1x10 7 to 1x1011CFU/mL, more preferably
8 1x10 to lxlO 0 CFU/mL with respect to the heated fermented
product. When the Bifidobacterium bifidum are dead cells,
CFU/mL may be replaced with individual cells/mL.
[0125]
After Bifidobacterium bifidum and human milk oligosaccharides
are added to the heated fermented product, concentration is
performed. The concentration process may be performed by
properly using a conventionally known concentration method.
In the centrifugalseparationmethod, wheyin the concentration
target (the heated fermented product to which Bifidobacteria
and prebiotics are added) is removed to obtain concentrated
fermented milk of Bifidobacterium bifidum in which the solid
content concentrationis increased. Ingestion of the obtained
fermented milk makes it possible to improve intestinal flora.
[0126]
<Production Example 5>
A method of producing modified milk powder to which
Bifidobacterium bifidum is added will be described below.
kg of desalted milk whey protein powder (manufactured by
66 17287359_1 (GHMatters) P114129.AU
Mirai), 6 kg of milk casein powder (manufactured by Fonterra),
48 kg of lactose (manufactured by Mirai), 920 g of a mineral
mixture (manufactured by Tomita Pharmaceutical Co., Ltd.), 32
g of a vitamin mixture (manufactured by Tanabe Seiyaku Co.,
Ltd.), human milk oligosaccharides, 500 g of lactulose
(manufactured by Morinaga Milk Industry Co., Ltd.), 500 g of
raffinose (manufactured by Nippon Beet Sugar Manufacturing Co.,
Ltd.), and 900 g of galactooligosaccharide liquid sugar
(manufactured by Yakult Pharmaceutical Industry Co., Ltd.) are
dissolved in 300 kg of warm water, and then dissolved at 900C
for 10 min through heating, and homogenized with addition of
28 kgofprepared fat (manufacturedbyTaiyoYushiCorp.). Then,
sterilization, and concentration steps are performed, and
about 95 kg of modified milk powder is prepared through
spray-drying. To this, 100 g of microbial cell powder of
Bifidobacterium bifidum, and 100 g of microbial cell powder
of lactobacilli/Bifidobacteria, which are triturated with
starch, are added to prepare about 95 kg ofmodifiedmilk powder
blended with Bifidobacterium bifidum/human milk
oligosaccharides (available from Funakoshi). When the
obtained modified milk powder is dissolved in water, and
becomes a milk preparation at a total solid content
concentration of 14% (w/V) (as a standard milk preparation
concentration), the number of bacteria of Bifidobacterium
9 bifidum in the milk preparation may become 2.7x10 CFU/100ml.
67 17287359_1 (GHMatters) P114129.AU
When the modified milk powder obtained as described above
is administered, proliferation of probiotics may be promoted
in the intestine.
[0127]
When the nutritional compositions produced in the above
described Production Examples 1 to 5 are administered,
probiotics can use HMOs and proliferate in the intestine,
which leads to improvement of intestinal flora.
[0128]
It is to be understood that, if any prior art publication
is referred to herein, such reference does not constitute
an admission that the publication forms a part of the common
general knowledge in the art, in Australia or any other
country.
[0129]
In the claims which follow and in the preceding description
of the invention, except where the context requires
otherwise due to express language or necessary implication,
the word "comprise" or variations such as "comprises" or
"comprising" is used in an inclusive sense, i.e. to specify
the presence of the stated features but not to preclude the
presence or addition of further features in various
embodiments of the invention.
68 18618821_1 (GHMatters) P114129.AU
JPOXMLDOC01‐seql (18).app SEQUENCE LISTING
<110> MORINAGA MILK INDUSTRY CO., LTD. <120> NUTRITION COMPOSITION, FOOD AND DRINK COMPOSITION AND MODIFIED MILK POWDER USING THE NUTRIENT COMPOSITION
<130> FPCT729
<150> JP2018‐065647 <151> 2018‐03‐29
<160> 22
<170> PatentIn version 3.5
<210> 1 <211> 21 <212> DNA <213> Artificial sequence
<220> <223> Forward primer of Bifidobacterium bifidum
<400> 1 ccacatgatc gcatgtgatt g 21
<210> 2 <211> 19 <212> DNA <213> Artificial sequence
<220> <223> Reverse primer of Bifidobacterium bifidum
<400> 2 ccgaaggctt gctcccaaa 19
<210> 3 <211> 20 <212> DNA <213> Artificial sequence
<220> <223> Forward primer of Bifidobacterium longum subsp longum
Page 1
JPOXMLDOC01‐seql (18).app <400> 3 ttccagttga tcgcatggtc 20
<210> 4 <211> 20 <212> DNA <213> Artificial sequence
<220> <223> Reverse primer of Bifidobacterium longum subsp longum
<400> 4 gggaagccgt atctctacga 20
<210> 5 <211> 19 <212> DNA <213> Artificial sequence
<220> <223> Forward primer of Bifidobacterium adlescentis
<400> 5 ctccagttgg atgcatgtc 19
<210> 6 <211> 18 <212> DNA <213> Artificial sequence
<220> <223> Reverse primer of Bifidobacterium adlescentis
<400> 6 cgaaggcttg ctcccagt 18
<210> 7 <211> 17 <212> DNA <213> Artificial sequence
<220> <223> Forward primer of Bifidobacterium catenulatum group
Page 2
JPOXMLDOC01‐seql (18).app <400> 7 cggatgctcc gactcct 17
<210> 8 <211> 18 <212> DNA <213> Artificial sequence
<220> <223> Reverse primer of Bifidobacterium catenulatum group
<400> 8 cgaaggcttg ctcccgat 18
<210> 9 <211> 18 <212> DNA <213> Artificial sequence
<220> <223> Forward primer of Bifidobacterium pseudolongum
<400> 9 cacatgagcg catgcgag 18
<210> 10 <211> 19 <212> DNA <213> Artificial sequence
<220> <223> Reverse primer of Bifidobacterium pseudolongum
<400> 10 tccactcaac acggccgaa 19
<210> 11 <211> 18 <212> DNA <213> Artificial sequence
<220> <223> Forward primer of Bifidobacterium animalis subsp lactis
Page 3
JPOXMLDOC01‐seql (18).app <400> 11 gtggagacac ggtttccc 18
<210> 12 <211> 20 <212> DNA <213> Artificial sequence
<220> <223> Reverse primer of Bifidobacterium animalis subsp lactis
<400> 12 cacaccacac aatccaatac 20
<210> 13 <211> 19 <212> DNA <213> Artificial sequence
<220> <223> Forward primer of Lactobacillus casei
<400> 13 cgagttctcg ttgatgatc 19
<210> 14 <211> 19 <212> DNA <213> Artificial sequence
<220> <223> Reverse primer of Lactobacillus casei
<400> 14 aagattccct actgctgcc 19
<210> 15 <211> 19 <212> DNA <213> Artificial sequence
<220> <223> Forward primer of Lactobacillus plantarum
Page 4
JPOXMLDOC01‐seql (18).app <400> 15 agatttgatc atggctcag 19
<210> 16 <211> 20 <212> DNA <213> Artificial sequence
<220> <223> Reverse primer of Lactobacillus plantarum
<400> 16 cggtattagc atctgtttcc 20
<210> 17 <211> 17 <212> DNA <213> Artificial sequence
<220> <223> Forward primer of Lactobacillus acidophilus
<400> 17 acagattcac ttcggtg 17
<210> 18 <211> 23 <212> DNA <213> Artificial sequence
<220> <223> Reverse primer of Lactobacillus acidophilus
<400> 18 aaaggccagt tactacctct atc 23
<210> 19 <211> 24 <212> DNA <213> Artificial sequence
<220> <223> Forward primer of Lactobacillus rhamnosus
Page 5
JPOXMLDOC01‐seql (18).app <400> 19 gcaagtcgaa cgagttctga ttat 24
<210> 20 <211> 26 <212> DNA <213> Artificial sequence
<220> <223> Reverse primer of Lactobacillus rhamnosus
<400> 20 gccgacaaca gttactctgc cgacca 26
<210> 21 <211> 23 <212> DNA <213> Artificial sequence
<220> <223> Forward primer of Lactobacillus gasseri
<400> 21 gatgaatttg gtgcttgcac cag 23
<210> 22 <211> 19 <212> DNA <213> Artificial sequence
<220> <223> Reverse primer of Lactobacillus gasseri
<400> 22 aagattccct actgctgcc 19
Page 6
Claims (3)
- [Claim 1]A nutritional composition containing:bacteria belonging to Bifidobacterium bifidum;one or more types of probiotics having a low abilityto assimilate human milk oligosaccharides; andhuman milk oligosaccharides,wherein the bacteria belonging to Bifidobacteriumbifidum are at least one bacterium selected fromBifidobacterium bifidum ATCC29521, Bifidobacterium bifidum(NITE BP-2429), Bifidobacterium bifidum (NITE BP-1252),Bifidobacterium bifidum (NITE BP-2431), Bifidobacteriumbifidum (NITE BP-02648), Bifidobacterium bifidum (NITE BP02432), and Bifidobacterium bifidum (NITE BP-02433),wherein the human milk oligosaccharides are 2'fucosyllactose and/or lacto-N-neotetraose,wherein the bacteria belonging to the Bifidobacteriumbifidum is Bifidobacterium bifidum that has been culturedin vitro,wherein, when the human milk oligosaccharide is 2'fucosyllactose, the probiotic is at least one bacteriumselected from: Bifidobacterium longum subspecies longum,Bifidobacterium pseudocatenulatum, Bifidobacterium animalissubspecies lactis, Lactobacillus paracasei, Lactobacillusacidophilus, Lactobacillus rhamnosus and Lactobacillus69 19016887_1 (GHMatters) P114129.AU gasseri, and wherein, when the human milk oligosaccharide is lactoN-neotetraose, the probiotic is at least one bacteriumselected from: Bifidobacterium longum subspecies longum,Lactobacillus paracasei, Lactobacillus plantarum,Lactobacillus acidophilus, and Lactobacillus rhamnosus.
- [Claim 2]A food/drink composition containing the nutritionalcomposition described in claim 1.
- [Claim 3]Modified milk powder containing the nutritionalcomposition described in claim 1.70 19016887_1 (GHMatters) P114129.AU
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| JP2018-065647 | 2018-03-29 | ||
| JP2018065647 | 2018-03-29 | ||
| PCT/JP2019/012894 WO2019189200A1 (en) | 2018-03-29 | 2019-03-26 | Nutritional composition, food/drink composition using nutritional composition, and modified milk powder using nutritional composition |
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| US (1) | US20210068414A1 (en) |
| EP (1) | EP3782480A4 (en) |
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| WO2021059894A1 (en) * | 2019-09-27 | 2021-04-01 | 森永乳業株式会社 | Composition, food/beverage composition that contains said composition, and modified milk |
| CN112806577B (en) * | 2019-11-15 | 2021-11-26 | 内蒙古伊利实业集团股份有限公司 | Prebiotic probiotic synergistic combinations for butyric acid production |
| JP7474596B2 (en) * | 2020-01-20 | 2024-04-25 | 森永乳業株式会社 | Novel Bifidobacterium bacteria, compositions containing said bacteria, and compositions for promoting the growth of said bacteria |
| US20240000868A1 (en) * | 2020-11-16 | 2024-01-04 | Inner Mongolia Yili Industrial Group Co., Ltd. | Composition Containing Bifidobacterium Lactis and Human Milk Oligosaccharides and Application Thereof |
| WO2022173764A1 (en) * | 2021-02-10 | 2022-08-18 | Winks Brands Inc. | Nutritional plant-based foods and beverages, methods of manufacture, and methods of treatment |
| JP6978621B1 (en) * | 2021-03-12 | 2021-12-08 | 森永乳業株式会社 | Composition |
| CN114806980B (en) * | 2022-06-29 | 2022-11-04 | 微康益生菌(苏州)股份有限公司 | Culture medium for culturing living biological medicine and application thereof |
| CN116491655A (en) * | 2023-04-21 | 2023-07-28 | 合生元（广州）健康产品有限公司 | Application of probiotic prebiotic composition in preparation of food for improving intestinal probiotic colonization |
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| CA2822500C (en) * | 2010-12-31 | 2022-07-26 | Abbott Laboratories | Human milk oligosaccharides to promote growth of beneficial bacteria |
| CN103763940B (en) * | 2010-12-31 | 2015-10-14 | 雅培制药有限公司 | Promote the probio of effective microorganisms growth and the conjunction benefit element combination of human milk oligosaccharides |
| EP2842560A4 (en) * | 2012-04-23 | 2015-12-02 | Univ Kyoto | Composition for promoting bifidobacteria growth |
| WO2014086373A1 (en) | 2012-12-07 | 2014-06-12 | Glycom A/S | Crystallisation of human milk oligosaccharides (hmo) |
| US10165788B2 (en) * | 2013-06-17 | 2019-01-01 | The Regents Of The University Of California | Methods and compositions for improved digestion of milk oligosaccharides |
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2019
- 2019-03-26 EP EP19775704.0A patent/EP3782480A4/en active Pending
- 2019-03-26 CN CN201980021823.3A patent/CN111935995A/en active Pending
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| EP3782480A4 (en) | 2022-01-12 |
| JPWO2019189200A1 (en) | 2021-04-15 |
| EP3782480A1 (en) | 2021-02-24 |
| US20210068414A1 (en) | 2021-03-11 |
| WO2019189200A1 (en) | 2019-10-03 |
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